miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

Development of interleukin-17-producing γδ T cells is restricted to a functional embryonic wave.

γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

Limited niche availability suppresses murine intrathymic dendritic-cell development from noncommitted progenitors.

The origins of dendritic cells (DCs) and other myeloid cells in the thymus have remained controversial. In this study, we assessed developmental relationships between thymic dendritic cells and thymocytes, employing retrovirus-based cellular barcoding and reporter mice, as well as intrathymic transfers coupled with DC depletion. We demonstrated that a subset of early T-lineage progenitors expressed CX3CR1, a bona fide marker for DC progenitors. However, intrathymic transfers into nonmanipulated mice, as well as retroviral barcoding, indicated that thymic dendritic cells and thymocytes were largely of distinct developmental origin. In contrast, intrathymic transfers after in vivo depletion of DCs resulted in intrathymic development of non-T-lineage cells. In conclusion, our data support a model in which the adoption of T-lineage fate by noncommitted progenitors at steady state is enforced by signals from the thymic microenvironment unless niches promoting alternative lineage fates become available.

Induced and thymus-derived Foxp3⁺ regulatory T cells share a common niche.

Foxp3⁺ regulatory T (Treg) cells, which play a central role for the maintenance of immune homeostasis and self-tolerance, are known to be both generated in the thymus (thymus-derived, tTreg cells) and in the periphery, where they are converted from conventional CD4⁺ T cells (induced Treg (iTreg) cells). Recent data suggest a division of labor between these two Treg-cell subsets since their combined action was shown to be essential for protection in inflammatory disease models. Here, using the transfer colitis model, we examined whether tTreg cells and iTreg cells fill different niches within the CD4⁺ T-cell compartment. When naive T cells were co-transferred with either pure tTreg cells or with a mixture of tTreg cells and iTreg cells, induction of Foxp3⁺ Treg cells from naive T cells was not hampered by preoccupation of the Treg-cell niche. Using neuropilin-1 (Nrp1) as a surface marker to separate tTreg cells and iTreg cells, we demonstrate that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and display comparable TCR repertoires. However, when transferred together Nrp1⁺ tTreg cells outcompeted Nrp1⁻ iTreg cells and dominated the Treg-cell compartment. Taken together, our data suggest that tTreg cells and iTreg cells share a common peripheral niche.

Differential postselection proliferation dynamics of αß T cells, Foxp3+ regulatory T cells, and invariant NKT cells monitored by genetic pulse labeling.

The thymus generates two divergent types of lymphocytes, innate and adaptive T cells. Innate T cells such as invariant NKT cells provide immediate immune defense, whereas adaptive T cells require a phase of expansion and functional differentiation outside the thymus. Naive adaptive T lymphocytes should not proliferate much after positive selection in the thymus to ensure a highly diverse TCR repertoire. In contrast, oligoclonal innate lymphocyte populations are efficiently expanded through intrathymic proliferation. For CD4(+)Foxp3(+) regulatory T cells (Tregs), which are thought to be generated by agonist recognition, it is not clear whether they proliferate upon thymic selection. In this study, we investigated thymic and peripheral T cell proliferation by genetic pulse labeling. To this end, we used a mouse model in which all developing αß thymocytes were marked by expression of a histone 2B-enhanced GFP (H2BeGFP) fusion-protein located within the Tcrd locus (TcrdH2BeGFP). This reporter gene was excised during TCR α-chain VJ-recombination, and the retained H2BeGFP signal was thus diluted upon cell proliferation. We found that innate T cells such as CD1d-restricted invariant NKT cells all underwent a phase of intense intrathymic proliferation, whereas adaptive CD4(+) and CD8(+) single-positive thymocytes including thymic Tregs cycled, on average, only once after final selection. After thymic exit, retention or loss of very stable H2BeGFP signal indicated the proliferative history of peripheral αß T cells. There, peripheral Tregs showed lower levels of H2BeGFP compared with CD4(+)Foxp3(-) T cells. This further supports the hypothesis that the Treg repertoire is shaped by self-Ag recognition in the steady-state.

Visualization and quantification of monoallelic TCRα gene rearrangement in αß T cells.

T-cell receptor α (TCRα) chain rearrangement is not constrained by allelic exclusion and thus αß T cells frequently have rearranged both alleles of this locus. Thereby, stepwise secondary rearrangements of both TCRα loci further increase the odds for generation of an α-chain that can be positively selected in combination with a pre-existing TCRß chain. Previous studies estimated that approximately 2-12% of murine and human αß T cells still carry one TCRα locus in germline configuration, which must comprise a partially or even fully rearranged TCRδ locus. However, these estimates are based on a relatively small amount of individual αß T-cell clones and αß T-cell hybridomas analyzed to date. To address this issue more accurately, we made use of a mouse model, in which a fluorescent reporter protein is introduced into the constant region of the TCRδ locus. In this TcrdH2BeGFP system, fluorescence emanating from retained TCRδ loci enabled us to quantify monoallelically rearranged αß T cells on a single-cell basis. Via fluorescence-activated cell sorting analysis, we determined the frequency of monoallelic TCRα rearrangements to be 1.7% in both peripheral CD4(+) and CD8(+) αß T cells. Furthermore, we found a skewed 5' Jα gene utilization of the rearranged TCRα allele in T cells with monoallelic TCRα rearrangements. This is in line with previous descriptions of a tight interallelic positional coincidence of Jα gene segments used on both TCRα alleles. Finally, analysis of T cells from transgenic mice harboring only one functional TCRα locus implied the existence of very rare unusual translocation or episomal reintegration events of formerly excised TCRδ loci.

IFN-γ production by allogeneic Foxp3+ regulatory T cells is essential for preventing experimental graft-versus-host disease.

It is emerging that CD4+Foxp3+ regulatory T (Treg) cells can produce the proinflammatory cytokine IFN-γ when stimulated in a Th1 cytokine environment. In this study, we report that Foxp3+ Treg cells readily produced IFN-γ in vivo in a highly inflammatory model of graft-versus-host disease (GVHD) and during a Th1-dominated immune response to intracellular bacteria. Moreover, stimulation in vitro via TCR in the presence of IL-12 alone was sufficient to induce IFN-γ production by Treg cells in a dose-dependent manner. Transfer of donor Treg cells can prevent lethal GVHD; therefore, we used this model as a robust readout for in vivo Treg function. Interestingly, >50% of allogeneic donor, but not residual recipient Foxp3+ Treg cells produced IFN-γ after transplantation, suggesting that this cytokine production was alloantigen specific. These IFN-γ producers were stable Foxp3+ Treg cells because methylation analysis of the Foxp3 gene locus of transferred and reisolated Treg cells during GVHD showed a fully demethylated Treg-specific-demethylated region. Next, we addressed whether IFN-γ production was supporting or rather impairing the immunosuppressive function of Treg cells during GVHD. Blocking of IFN-γ with specific mAb completely abolished the beneficial effect of donor Treg cells. We could further show that only wild-type Treg cells, but not Treg cells from IFN-γ-deficient donor mice, prevented GVHD. This indicated that Treg cell-intrinsic IFN-γ production was required for their protective function. In conclusion, our data show that IFN-γ produced by Foxp3+ Treg cells has essential immune-regulatory functions that are required for prevention of experimental GVHD.

High TCR diversity ensures optimal function and homeostasis of Foxp3+ regulatory T cells.

Dominant tolerance to self-antigen requires the presence of sufficient numbers of CD4(+) Foxp3(+) Treg cells with matching antigen specificity. However, the size and role of TCR repertoire diversity for antigen-specific immuno-regulation through Treg cells is not clear. Here, we developed and applied a novel high-throughput (HT) TCR sequencing approach to analyze the TCR repertoire of Treg cells and revealed the importance of high diversity for Treg-cell homeostasis and function. We found that highly polyclonal Treg cells from WT mice vigorously expanded after adoptive transfer into non-lymphopenic TCR-transgenic recipients with low Treg-cell diversity. In that system, we identified specific Treg-cell TCR preferences in distinct anatomic locations such as the mesenteric LN indicating that Treg cells continuously compete for MHC class-II-presented self-, food-, or flora-antigen. Functionally, we showed that high TCR diversity was required for optimal suppressive function of Treg cells in experimental acute graft versus host disease (GvHD). In conclusion, we suggest that efficient immuno-regulation by Treg cells requires high TCR diversity. Thereby, continuous competition of peripheral Treg cells for limited self-antigen shapes an organ-optimized, yet highly diverse, local TCR repertoire.

CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells.

Gammadelta T cells are a potent source of innate IL-17A and IFN-gamma, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24(low) CD44(high) effector gammadelta T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ gammadelta T cells produced IL-17A, while NK1.1+ gammadelta T cells were efficient producers of IFN-gamma but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ gammadelta T cells. Accordingly, both gammadelta T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-gamma in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-gamma and IL-23 induced IL-17A production by NK1.1+ or CCR6+ gammadelta T cells, respectively. Importantly, we show that CCR6+ gammadelta T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL-17A-producing gammadelta T cells derive from less TCR-dependent selection events than IFN-gamma-producing NK1.1+ gammadelta T cells.

A clonotypic Vγ4Jγ1/Vδ5Dδ2Jδ1 innate γδ T-cell population restricted to the CCR6⁺CD27⁻ subset.

Here we investigate the TCR repertoire of mouse Vγ4(+) γδ T cells in correlation with their developmental origin and homeostasis. By deep sequencing we identify a high frequency of straight Vδ5Dδ2Jδ1 germline rearrangements without P- and N-nucleotides within the otherwise highly diverse Trd repertoire of Vγ4(+) cells. This sequence is infrequent in CCR6(-)CD27(+) cells, but abundant among CCR6(+)CD27(-) γδ T cells. Using an inducible Rag1 knock-in mouse model, we show that γδ T cells generated in the adult thymus rarely contain this germline-rearranged Vδ5Dδ2Jδ1 sequence, confirming its fetal origin. Single-cell analysis and deep sequencing of the Trg locus reveal a dominant CDR3 junctional motif that completes the TCR repertoire of invariant Vγ4(+)Vδ5(+) cells. In conclusion, this study identifies an innate subset of fetal thymus-derived γδ T cells with an invariant Vγ4(+)Vδ5(+) TCR that is restricted to the CCR6(+)CD27(-) subset of γδ T cells.

Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine.

Intestinal immunoglobulin A (IgA) ensures host defense and symbiosis with our commensal microbiota. Yet previous studies hint at a surprisingly low diversity of intestinal IgA, and it is unknown to what extent the diverse Ig arsenal generated by somatic recombination and diversification is actually used. In this study, we analyze more than one million mouse IgA sequences to describe the shaping of the intestinal IgA repertoire, its determinants, and stability over time. We show that expanded and infrequent clones combine to form highly diverse polyclonal IgA repertoires with very little overlap between individual mice. Selective homing allows expanded clones to evenly seed the small but not large intestine. Repertoire diversity increases during aging in a dual process. On the one hand, microbiota-, T cell-, and transcription factor RORγt-dependent but Peyer's patch-independent somatic mutations drive the diversification of expanded clones, and on the other hand, new clones are introduced into the repertoire of aged mice. An individual's IgA repertoire is stable and recalled after plasma cell depletion, which is indicative of functional memory. These data provide a conceptual framework to understand the dynamic changes in the IgA repertoires to match environmental and intrinsic stimuli.

BRCA1 and BRCA2 heterozygosity in embryonic stem cells reduces radiation-induced Rad51 focus formation but is not associated with radiosensitivity.

PURPOSE: The breast cancer susceptibility genes BRCA1 (breast cancer 1) and BRCA2 (breast cancer 2) encode proteins involved in double-strand break (DSB) repair, whose functions include facilitating homologous recombination through interactions with Rad51, the human homologue of bacterial RecA. Homozygous deficiency inhibits Rad51 focus formation and enhances radiosensitivity, but the effects of heterozygosity have not been investigated in detail. The purpose of this work was to examine the effect of heterozygosity on Rad51 activation and clonogenicity following X-irradiation (XR). MATERIALS AND METHODS: We used quantitative assessment of immunofluorescent foci to assess Rad51 activation in wild type mouse embryonic fibroblasts (MEF) and in paired mutant and wild type BRCA1 and BRCA2 embryonic stem cells (ES cells). We measured radiosensitivity in the same cell lines using clonogenic survival assays. RESULTS: ES cells exhibit higher numbers of cells with Rad51 foci post radiation than MEF, likely due to differences in cell cycle distribution. Compared to wild type cells, BRCA1 and BRCA2 heterozygous ES cells demonstrate lower numbers of Rad51 foci per nucleus 4 and 24 hours post radiation. This was not associated with significantly enhanced radiosensitivity. CONCLUSIONS: BRCA1/2 heterozygosity in ES cells is associated with a subtle reduction in Rad51 foci formation that is not associated with increased XR induced cytotoxicity.

Tspy is nonfunctional in the Mongolian gerbil but functional in the Syrian hamster.

The TSPY gene is conserved in placental mammals and encodes the testis-specific protein, Y encoded. Within the testis, TSPY expression is restricted to germ cells, and it is assumed that TSPY plays a role in the proliferation of germ cells. Since it was first discovered in humans, TSPY orthologous gene families have been subsequently characterized in many mammalian lineages. In contrast to the situation in cattle and primates, in which TSPY is organized in a moderately repetitive cluster, including functional members and pseudogenes, a peculiar situation is observed in rodents, in which Tspy has been become low or single copy and degenerated to a pseudogene in some species of the subgenus Mus. We have extended this approach and investigated Tspy gene evolution in the Syrian hamster (Mesocricetus auratus) and the Mongolian gerbil (Meriones unguiculatus). Whereas the Syrian hamster Tspy is functionally conserved, organized in multiple copies, and expressed only in testis, the closely related Mongolian gerbil possesses a single-copy pseudogene that is unable to generate a functional transcript. Thus, the Tspy locus has degenerated at least twice at different points of rodent evolution, strongly supporting the hypothesis that the decay of Y-chromosomal genes is an intrinsic evolutionary process. TSPY is the first example of a Y-chromosomal tandem repetitive gene whose decay could be studied in two independent mammalian lineages.